28th June 2018
Peptidome
The set of peptides presented by class I MHC molecules at the cell surface.
Hypotheses for the role of MHC I in skin sensitisation
- Sensitiser-modified peptides presented by MHC I directly stimulate the immune system.
- Sensitiser modification of proteins alters the MHC I presented reportoire of peptides.
HaCaTs and DNCB
- HaCaT cell line is a spontaneously transformed keratinocyte.
- Homozygous HLA-A*31:01, HLA-B*40:01 / HLA-B*51:01, HLA-C*03 / HLA-C*15
- 2,4-Dinitrochlorobenzene (DNCB) is our model sensitiser.
- DNCB modifies 0.12% Lys, 0.03% Cys and < 0.03% Tyr, His and Arg.
HaCaT peptidome
Wildtype
HaCaT N-mer peptide counts
HaCaT N-mer peptide frequency
Immune epitope database peptides
HaCaT predicted peptides
DNCB modified peptides
| Peptide | Length | Replicate | UNIPROT ID |
|---|---|---|---|
| AETEC(+166.00)RYAL | 9 | DNCB 1A | P13646 |
| RFC(+166.00)PFAERTR | 10 | DNCB 1A | P78417 |
| RFC(+166.00)PFAERTR | 10 | DNCB 1B | P78417 |
| AETEC(+166.00)RYAL | 9 | DNCB 1B | P13646 |
| AETEC(+166.00)RYAL | 9 | DNCB 3A | P13646 |
| RFC(+166.00)PFAERTR | 10 | DNCB 3A | P78417 |
| AETEC(+166.00)RYAL | 9 | DNCB 3B | P13646 |
HLA-B*40:01 9-mer AETEC(+DNP)RYAL
Keratin, type I cytoskeletal 13
HLA-A*31:01 10-mer RFC(+DNP)PFAERTR
Glutathione S-transferase omega-1
Glutathione S-transferase omega-1
Next steps
- HLA-B*40:01, 9mer, AETECRYAL, Keratin, type I cytoskeletal 13
- HLA-A*31:01, 10mer, RFCPFAERTR, Glutathione S-transferase omega-1
- HLA-A*02:01, 9mer, YSMRFCPFA, Glutathione S-transferase omega-1
Erika's observes DNCB modified tryptic peptides :
- MFAPTK(+DNP)TWR, HLA-A*31:01, 9mer, MFAPTKTWR, 60S ribosomal protein L4
- YFDK(+DNP)ASYR, HLA-A*31:01, 9mer, KYFDKASYR, Transketolase
Erika also observed MCI modified Glutathione S-transferase P:
- PPYTVVY(+MCI)FPVR, HLA-A*31:01 9-mer, VVYFPVRGR
HaCaT peptidome
HLA-A*02:01 transfectant
HLA-A2 transfectant N-mer peptide counts
HLA-A2 transfectant N-mer peptide frequency
Immune epitope database peptides
Machine Learning classified peptides
Predicted HLA-A2 peptides
P13646: Keratin type I cytoskeletal 13, only seen as unmodified peptides
| Peptide | Length | Replicate | UNIPROT ID |
|---|---|---|---|
| MLLDIKTRL | 9 | Control 1 | P13646 |
| MLLDIKTRL | 9 | Control 2 | P13646 |
| RLASYLEKV | 9 | Control 2 | P13646 |
| MLLDIKTRL | 9 | Control 3 | P13646 |
| MLLDIKTRL | 9 | DNCB 1 | P13646 |
| MLLDIKTRL | 9 | DNCB 2 | P13646 |
Glutathione S-transferase Mu 4
HLA-A31 peptide also seen in WT assays
| Peptide | Length | Allotype | UNIPROT ID |
|---|---|---|---|
| FLGKRPWFV | 9 | HLA-A2 | Q03013 |
| RFLPKPLYTR | 10 | HLA-A31 | Q03013 |
DNCB modified HLA-A2 peptides
P13647 : Keratin, type II cytoskeletal 5
P13489 : Ribonuclease inhibitor
| Peptide | Length | Replicate | UNIPROT ID |
|---|---|---|---|
| KLLEGEEC(+166.00)RL | 10 | DNCB 2 | P13647 |
| GLLDPQC(+166.00)RL | 9 | DNCB 2 | P13489 |
| KLLEGEEC(+166.00)RL | 10 | DNCB 3 | P13647 |
HLA-A*02:01 9-mer, GLLDPQC(+DNP)RL
Ribonuclease inhibitor
Ribonuclease inhibitor
HLA-A*02:01 10-mer, KLLEGEEC(+DNP)RL
Keratin, type II cytoskeletal 5
Story so far
Evidence for direct presentation of sensitiser modified peptides:
- Peptides identified by mass spectrometry
- Peptides predicted to be strong binders
- Observing modified peptides from several proteins is not inconsistent with the ability of DNCB to induce contact hypersensitivity in all immunocompetent individuals
Evidence for sensitiser effect on cellular detoxification mechanism:
- Modification of Glutathione S-transferase omega-1
Next steps
- Synthesise peptides to verify spectra and test immunogenecity.
- Process and analyse HaCaT protein turnover data.
- HaCaT A2-transfectant protein turnover.
- Look for further evidence of presentation of peptides from proteins associated with oxidative stress